In 1975, it was reported that individual, normal antibody-secreting cells could be fused with myeloma cells to produce continuous cell lines which stably secreted monoclonal antibodies (G. Kohler et al. (1975) “Continuous cultures of fused cells secreting antibody of predefined specificity”, Nature 256: 495-497). Since then, numerous publications and patents have described the production and use of monoclonal antibodies to diverse antigens. See the following reviews for more details. (E. M. Yoo et al. (2002) “Myeloma expression systems”, Journal of Immunological Methods 261: 1-20; P. N. Nelson et al. (2000) “Monoclonal Antibodies”, Molecular Pathology 53: 111-117).
In monoclonal antibody production, each hybridoma cell synthesizes a homogeneous or monoclonal immunoglobulin population that represents one of many antibodies which are produced by the spleen of the immunized animals used to create the hybridoma cell. For example, U.S. Pat. No. 5,055,405 discloses the production of hybridoma cell lines which produce antibodies against the periodontal pathogen, Treponema denticola. And U.S. Pat. No. 6,818,215 discloses the production of hybridoma cell lines which produce antibodies against senescent cell-derived inhibitors of DNA synthesis. The present invention discloses antibodies specific to Prox1 from diverse vertebrate species.
Mouse Prox1 was first identified in 1993 as a vertebrate gene related to the Drosophila protein Prospero (G. Oliver, et al. (1993) “Prox1, a Prospero-related homeobox gene expressed during murine development”, Mechanisms of Development 44: 3-16). Subsequently, Prox1 genes were found in diverse vertebrates including chicken, human and newts. Deletion of the Prox1 gene from the mouse genome showed that it was essential for development of the eye, liver, and lymphatic system (J. T. Wigle et al. (1999) “Prox1 function is crucial for mouse lens-fiber elongation”, Nat. Genet. 21: 318-322). Rabbit polyclonal antibodies specific to human Prox1 were developed and used to detect this protein in the developing eye (M. K. Duncan et al. (2002) “Prox1 is differentially localized during lens development”, Mech. Dev. 112: 195-198). Subsequently, Prox1 was shown to be an excellent marker to differentiate lymphatic vessels from blood vessels in vivo (Y. K. Hong et al. (2002) “Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate”, Dev. Dyn. 225: 351-357). Since many cancers metastasize to lymph nodes, some investigators have reported the presence of lymphatic vessels as predictive of the ability of a tumor to metastasize.
Prox1 is the vertebrate homolog of Prospero, a divergent homeodomain protein important for neuroblast fate determination and photoreceptor development in Drosophila (C. Q. Doe et al. (1991) “The Prospero gene specifies cell fates in the Drosophila central nervous system”, Cell 65: 451-464; B. Hassan et al. (1997) “Prospero is a panneural transcription factor that modulates homeodomain protein activity”, Proc. Natl. Acad. Sci. USA 94: 10991-10996; T. Cook et al. (2003) “Distinction between color photoreceptor cell fates is controlled by Prospero in Drosophila”, Dev. Cell 4: 853-864). Human Prox1 is a 736 amino acid protein containing an N-terminal nuclear localization signal, three nuclear receptor boxes, a nuclear export signal and a highly conserved C-terminus containing the divergent homeodomain and the novel Prospero domain (T. R. Burglin (1994) “A Caehorhabditis elegans homologue defines a novel domain”, Trends Biochem. Sci. 19: 70-71; S. I. Tomarev et al. (1998) “Characterization of the mouse Prox1 gene”, Biochem. Biophys. Res. Commun. 248: 684-689; J. Qin et al. (2004) “Prospero-related homeobox (Prox1) is a corepressor of human liver receptor homolog-1 and suppresses the transcription of the cholesterol 7-alpha-hydroxylase gene”, Mol. Endocrinol. 18: 2424-2439). Prox1 is highly expressed in lens fiber cells (M. K. Duncan et al. (2002) “Prox1 is differentially localized during lens development”, Mech. Dev. 112: 195-198) and Prox1 null mice are defective in lens fiber cell elongation (J. T. Wigle et al. (1999) “Prox1 function is crucial for mouse lens-fibre elongation”, Nat. Genet. 21: 318-322) and the differentiation of retinal horizontal cells (M. A. Dyer et al. (2003) “Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina”, Nat. Genet. 34: 53-58). In this context, Prox1 functions as a transcription factor and has been shown to transactivate both the chicken βB1- and mouse γF-crystallin promoters (J. Lengler et al. (2001) “Antagonistic action of six3 and prox1 at the gamma-crystallin promoter”, Nucleic Acids Res. 29: 515-526; W. Cui et al. (2004) “Mafs, Prox1 and Pax6 can regulate chicken beta B1-crystallin gene expression”, J. Biol. Chem. 279: 11088-11095).
While Prox1 is critical for eye development, the correct dosage of this protein is essential for embryogenesis since heterozygous Prox 1 null mice die shortly after birth on most genetic backgrounds while homozygous Prox1 nulls die at 14.5 dpc (J. T. Wigle et al. (1999) “Prox1 function is crucial for mouse lens-fibre elongation”, Nat. Genet. 21: 318-322; J. T. Wigle et al. (1999) “Prox1 function is required for the development of the murine lymphatic system”, Cell 98: 769-778). Analysis of these animals has shown that Prox1 is essential for the delamination of hepatocytes from the liver bud into the surrounding mesenchyme which is necessary for normal liver development (B. Sosa-Pineda et al. (2000) “Hepatocyte migration during liver development requires Prox1”, Nat. Genet. 25: 254-255). Expression studies have shown Prox1 to be one of the earliest molecular markers of liver/pancreatic fated ventral foregut endoderm (Z. Burke et al. (2002) “Prox1 is an early specific marker for the developing liver and pancreas in the mammalian foregut endoderm”, Mech. Dev. 118: 147-155). Prox1 expression is maintained in hepatoblasts and hepatocytes throughout development and is highly upregulated in transformed hepatoma cell lines (J. Dudas et al. (2004) “The homeobox transcription factor Prox1 is highly conserved in embryonic hepatoblasts and in adult and transformed hepatocytes, but is absent from bile duct epithelium”, Anat. Embryol. (Berl) 208: 359-366). Prox1 interacts with liver receptor homolog-1 (LRH-1), a transcription factor essential for the expression of enzymes important for bile acid synthesis, repressing LRH-1 transcriptional activity by impairing its binding to DNA (J. Qin et al. (2004) “Prospero-related homeobox (Prox1) is a corepressor of human liver receptor homolog-1 and suppresses the transcription of the cholesterol 7-alpha-hydroxylase gene”, Mol. Endocrinol. 18: 2424-2439).
Prox 1 is also a key player in the formation of the lymphatic system (Y. K. Hong et al. (2002) “Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate”, Dev. Dyn. 225: 351-357). Expression of Prox1 in a subpopulation of venous endothelial cells is one of the first indications that lymphangiogenesis has been initiated and cells biased to a lymphatic phenotype (J. T. Wigle et al. (2002) “An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype”, Embo. J 21: 1505-1513). Prox1 null mice do not develop lymphatics due to arrested endothelial budding from the primary vascular network (J. T. Wigle et al. (1999) “Prox1 function is required for the development of the murine lymphatic system”, Cell 98: 769-778). Overexpression of Prox1 can reprogram blood vascular endothelial cells into lymphatic endothelial cells confirming the central role of Prox1 in lymphatic specification (T. V. Petrova et al. (2002) “Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor”, Embo J. 21: 4593-4599). Immunostaining of lymphatic tissues from healthy human adults and lymphedema patients with Prox1 polyclonal antibodies showed that Prox1 is a reliable and highly specific marker for lymphatic endothelial cells in normal and pathologic human tissues (J. Wilting et al. (2002) “The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues”, Faseb J. 16: 1271-1273; J. S. Reis-Filho et al. (2003) “Lymphangiogenesis in tumors: what do we know?”, Microsc. Res. Tech. 60: 171-180; I. Van der Auwera et al. (2004) “Increased angiogenesis and lymphangiogenesis in inflammatory versus noninflammatory breast cancer by real-time reverse transcriptase-PCR gene expression quantification”, Clin. Cancer Res. 10: 7965-7971; M. A. Al-Rawi et al. (2005) “Lymphangiogenesis and its role in cancer”, Histol. Histopathol. 20: 283-298). However, the routine use of Prox1 staining in the clinic to identify lymphatics in biopsy specimens is impeded by the lack of highly standardized and reproducible anti-Prox1 monoclonal antibodies. In accordance with the invention, anti-Prox1 monoclonal antibodies are provided that can be used for Prox1 immunodetection in diverse vertebrates from humans to reptiles.